Automatic aseptic sampling valve for sampling from enclosed containers

ABSTRACT

A sample can be collected from an enclosed container by opening a sample collection valve and drawing the sample from the enclosed container. After delivery of the sample out of a fluid flow path, a sanitizing fluid can be directed along the fluid flow path to sanitize the fluid flow path.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No.PCT/US2012/036652, filed May 4, 2012, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of U.S.Provisional Patent Application No. 61/483,559, filed on May 6, 2011,U.S. Provisional Patent Application No. 61/488,627, filed on May 20,2011, and U.S. Provisional Patent Application No. 61/584,189, filed onJan. 6, 2012, each of which are incorporated herein by reference intheir entirety.

FIELD

The present disclosure is directed to an automatic aseptic samplingvalve and methods of using the same.

BACKGROUND

Obtaining samples from containers or other systems that supportbiologically and/or chemically active environments can require complexand careful sampling procedures to avoid contamination of the containersor the environment itself. For example, most bioreactors requirefrequent sampling (e.g., one or more times a day) to monitor and controlthe conditions and levels of nutrients needed for cell growth. To reducethe risk of contamination within such systems, conventional samplingtechniques generally require operators to perform multiple,labor-intensive steps.

SUMMARY

In some embodiments, the sampling systems and methods disclosed hereinprovide consistent sampling procedures for obtaining samples of adesired quality, while reducing the risk of contamination of thebioreactor and the need for labor-intensive operator attention.

A sampling system for collecting a fluid sample from an enclosedcontainer is provided. The system can include (a) a sanitizing fluidinlet valve operable between an open position and a closed position; (b)a gas inlet valve operable between an open position and a closedposition; (c) a sample collection valve operable between an openposition and a closed position; (d) an outlet valve operable between anopen position and a closed position; (e) a variable volume reservoir;and (f) a fluid flow path interconnecting (a)-(e). When (a), (b), and(d) are in the closed position, (c) can be in the open position towithdraw a sample from the enclosed container into the reservoir along afirst portion of the fluid flow path. When (a), (b), and (c) are in theclosed position, the sample can be discharged from the reservoir along asecond portion of the fluid flow path through (d). When (a) is in theopen position and (b) and (c) are in the closed position, a sanitizingfluid can be introduced into the fluid flow path through (a) to sanitizeat least the first portion of the fluid flow path.

In some embodiments, when (a) is in the open position, and (b) and (c)are in the closed position, the sanitizing fluid also sanitizes thereservoir. In other embodiments, (a) is at an upstream portion of thefluid flow path and (d) is at a downstream portion of the fluid flowpath, and the sanitizing fluid can flow through the fluid flow path from(a) to (d) to sanitize the fluid flow path between (a) and (d). In otherembodiments, (a)-(e) are interconnected along the fluid flow path fromthe upstream portion to the downstream portion in the following order:(a), (b), (c), (e), and (d). The reservoir can include a pump that isconfigured to draw the sample into the reservoir through a reservoirinlet and direct the sample out of the reservoir through a reservoiroutlet. The reservoir can include a diaphragm pump or other variablevolume pump that can be used to result in a positive displacement of asample, such as a syringe pump.

In other embodiments, a second outlet valve can be provided, with thesecond outlet valve being located downstream of the first outlet valve.When (a) is in the open position and (b), (c), and (d) are in the openposition, the sanitizing fluid can flow along the fluid flow pathbetween (a) and the second outlet valve to sanitize portions of thefluid flow path in the vicinity of (c) and (d). The second outlet valvecan be a variable back-pressure regulator. In some embodiments, thesecond outlet valve is a thermostatically-controlled valve.

In other embodiments, when (a) and (c) are in the closed position, and(b), (d), and the second outlet valve are in the open position, gas canbe introduced into the fluid flow path through (b) to purge thesanitizing fluid from at least the first and second portions of thefluid flow path. In some embodiments, the gas can function to cool thevalve in a case where the sanitizing fluid is hot (e.g., steam). Thesample collection valve can include a valve stem with a tapered sealingmember. A portion of the valve stem can extend into the fluid flow pathwhen the sample collection valve is in the closed position, such thatsanitizing fluid introduced into the fluid flow path by the sanitizingfluid inlet valve will flow past the portion of the valve stem thatextends into the fluid flow path.

In another embodiment, a method of collecting a fluid sample from anenclosed container is provided. The method can include opening asanitizing fluid inlet valve and directing sanitizing fluid downstreamthrough a fluid flow path past a closed sample collection valve and anopen first outlet valve, and discharging the sanitizing fluid out asecond outlet valve, with the second outlet valve being locateddownstream of the first outlet valve. A sample collection valve can beopened while the sanitizing fluid inlet valve and first outlet valve(and the gas inlet valve) are closed and a fluid sample can be drawnfrom the enclosed container into a variable volume reservoir along afirst portion of the fluid flow path. The fluid sample can be directedout of the reservoir along a second portion of the fluid flow path anddischarged out of the first outlet valve while the sanitizing fluidinlet valve and sample collection valve are closed. For a long distanceembodiment, air can be pumped following the sample, allowing arelatively small volume sample to be pumped long distances.

In some embodiments, after discharging the sanitizing fluid but beforedrawing the fluid sample, a gas inlet valve is opened and a gas isdirected downstream through the fluid flow path past the closed samplecollection valve and through the first open outlet valve. The gas can bedischarged through the second outlet valve to purge the sanitizing fluidfrom at least the first and second portions of the fluid flow path. Thereservoir can include a pump that is configured to draw the sample intothe reservoir through a reservoir inlet and direct the sample out of thereservoir through a reservoir outlet. In other embodiments, the pump canbe a diaphragm pump, and the sanitizing fluid can include steam.

In another embodiment, a method of collecting a sample from an enclosedcontainer is provided. The method can include directing a sanitizingfluid through a fluid flow path to sanitize or sterilize the fluid flowpath. The fluid flow path can have a gas inlet port downstream of thesanitizing fluid inlet, a sample inlet port downstream of the gas inletport, and a sample dispensing port downstream of the sample inlet port.The sanitizing fluid can be directed through the fluid flow path whilethe sample dispensing port is closed, and the sanitizing fluid can beexhausted through a control valve. Gas can be directed through the gasinlet port and into the fluid flow path while the sample dispensing portis closed. The gas can be exhausted through the control valve. A samplecan be drawn into the fluid flow path from the enclosed containerthrough the sample inlet port, and the sample can be dispensed out ofthe fluid flow path through the sample dispensing port. Additionalsanitizing fluid can be directed through the fluid flow path tore-sanitize or re-sterilize the fluid flow path while the sampledispensing port is closed.

In some embodiments, drawing and dispensing the sample comprisesactivating a variable volume reservoir to draw at least a portion of thesample into a chamber of the variable volume reservoir and dispense theportion of the sample from the chamber of the variable volume reservoirto the sample dispensing port. In other embodiments, a back pressure canbe provided by the control valve while the sanitizing fluid is directedthrough the fluid flow path to sanitize or sterilize the fluid flowpath. The control valve can include a diaphragm valve and the backpressure can be provided by increasing air pressure on the diaphragmvalve. In some embodiments, the control valve can direct sample to anend receiver/analyzer. The closure of the sample inlet port can includemoving a sealing tip of a valve stem so that the sealing tip engageswith the sample inlet port. When the sealing tip is engaged with thesample inlet port, at least a portion of the valve stem can extend intothe fluid flow path.

In some embodiments, the sampling system is made using materials thathave low heat transfer coefficients. In some embodiments, the samplingsystem is made using polymeric materials, such as thermoplastics andthermosetting materials. In some embodiments, the sampling system ismade using composite materials. In some embodiments, the sampling systemis formed by injection molding. In some embodiments, the sampling systemis formed by machining and drilling.

In some embodiments, the sampling system is modular in design, allowingselection of appropriate fittings for connecting to a wide variety ofapparatuses. In some embodiments, the variable volume reservoir ismodular, allowing selection of a reservoir suitable for the amount ofsample to be withdrawn from the enclosed container. In some embodimentsthe sampling system is compact to (1) reduce the hold-up volume of thesampling system, (2) allow rapid sanitizing of the sampling system, (3)allow for rapid removal of a sample from the enclosed container, or (4)any combination of (1), (2), or (3).

In some embodiments, a sample tube can dip down into the reactor fromoverhead allowing for the sampling into reactors above the liquid levelin the container. This arrangement can be particularly helpful in aprocess development scale reactor.

In some embodiments, the variable volume reservoir is designed tominimize the volume of gas that remains in the sampling system afterdischarge from the sampling system. In some embodiments, the ratio ofthe sample volume collected to the hold-up volume of the sampling systemis greater than 10:1, greater than 20:1, or even greater than 50:1.

In some embodiments, the variable volume reservoir is designed to pushthe sample collected out of the reservoir using a working fluid. In someembodiments, a positive pressure can be used via a working fluid. Inother embodiments, the system can create the positive pressure without aworking fluid, such as by using a syringe pump. In some embodiments, thepressure in the feed tank can be used to fill the reservoir, which ishooked to a piston—which can be pressurized (air or hydraulic fluid) todischarge the sample from the reservoir.

In some embodiments, the variable volume reservoir is designed to pullthe sample from the enclosed container into the reservoir by applying anegative pressure on the variable volume reservoir. This can beparticularly useful in systems with a draw tube from the top of thereactor—especially for small volume or development/experimentalreactors, which may not have a port located on the bottom of thebioreactor.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic view of a sampling system for obtainingsamples from enclosed containers.

FIGS. 2A-2D illustrate schematic views of a system for obtaining samplesfrom enclosed containers.

FIGS. 3A-3F illustrate a variable volume reservoir for drawing anddelivery samples from enclosed containers.

FIGS. 4A-4D illustrate schematic views of another system for obtainingsamples from enclosed containers.

FIGS. 5A-5C illustrate enlarged views of exemplary valves that can beused with a sampling system.

FIG. 6 illustrates a cross-sectional view of a system for obtainingsamples from enclosed containers.

FIG. 7 illustrates a partial cross-sectional view of a system forobtaining samples from enclosed containers.

FIG. 8 is an enlarged view of a portion of the system shown in FIG. 6.

FIG. 9 illustrates another embodiment of a system for obtaining samplesfrom enclosed containers.

FIG. 10 illustrates a partial view of a portion of a system forobtaining samples from enclosed containers.

FIG. 11 illustrates a control valve for use with a system for obtainingsamples from enclosed containers.

FIG. 12 illustrates another view of the control valve of FIG. 10.

FIGS. 13A-13D illustrate various views of another embodiment of a systemfor obtaining samples from enclosed containers.

FIG. 14 illustrates a variable volume reservoir that comprises asyringe-type device.

FIG. 15 illustrates another control valve for use with a system forobtaining samples from enclosed containers.

FIG. 16 illustrates a cross-sectional view of the valve shown in FIG.15.

FIG. 17 illustrates a cross-sectional view of the valve shown in FIG.15.

FIG. 18 illustrates the control valve of FIG. 15 shown from a liquidside.

FIG. 19 illustrates a cross-sectional view of the valve of FIG. 15.

FIG. 20 illustrates the control valve of FIG. 15 shown from an air side.

FIG. 21 illustrates a cross-sectional view of the valve of FIG. 15.

FIG. 22 illustrates a cross-sectional view of the valve of FIG. 15.

FIG. 23 illustrates a schematic view of an exemplary sampling system forobtaining samples from enclosed containers.

FIG. 24 illustrates a schematic view of an exemplary sampling system forobtaining samples from enclosed containers.

FIG. 25 illustrates a schematic view of an exemplary sampling system forobtaining samples from enclosed containers.

FIG. 26 illustrates a schematic view of an exemplary sampling system forobtaining samples from enclosed containers.

DETAILED DESCRIPTION

Various embodiments of sampling systems and their methods of use aredisclosed herein. The following description is exemplary in nature andis not intended to limit the scope, applicability, or configuration ofthe invention in any way. Various changes to the described embodimentmay be made in the function and arrangement of the elements describedherein without departing from the scope of the invention.

As used in this application and in the claims, the singular forms “a,”“an,” and “the” include the plural forms unless the context clearlydictates otherwise. Additionally, the term “includes” means “comprises.”Further, the term “coupled” generally means electrically,electromagnetically, and/or physically (e.g., mechanically orchemically) coupled or linked and does not exclude the presence ofintermediate elements between the coupled or associated items absentspecific contrary language.

The terms “upstream” and “downstream” are not absolute terms; instead,those terms refer to the direction of flow of fluids within a channel orpathway. Thus, with regard to a structure through which a fluid flows, afirst area is “upstream” of a second area if the fluid flows from thefirst area to the second area. Likewise, the second area can beconsidered “downstream” of the first area.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, percentages,measurements, distances, ratios, and so forth, as used in thespecification or claims are to be understood as being modified by theterm “about.” Accordingly, unless otherwise indicated, implicitly orexplicitly, the numerical parameters set forth are approximations thatmay depend on the desired properties sought and/or limits of detectionunder standard test conditions/methods. When directly and explicitlydistinguishing embodiments from discussed prior art, the embodimentnumbers are not approximates unless the word “about” is recited.

Although the operations of exemplary embodiments of the disclosed methodmay be described in a particular, sequential order for convenientpresentation, it should be understood that disclosed embodiments canencompass an order of operations other than the particular, sequentialorder disclosed. For example, operations described sequentially may insome cases be rearranged or performed concurrently. Further,descriptions and disclosures provided in association with one particularembodiment are not limited to that embodiment, and may be applied to anyembodiment disclosed.

FIG. 1 illustrates a sampling system 100 for obtaining a sample from abioreactor 102 or other similar containers or systems that supportbiologically and/or chemically active environments. Sampling system 100includes a sample collection valve 104 that can open to allow a sampleto enter a fluid flow path 106. The sample can be delivered along theflow path 106 to an outlet valve 108. Outlet valve 108 can open or closeto allow or restrict, respectively, the flow of samples through outletvalve 108. After the sample exits outlet valve 108, the sample can bedirected into an isolated chamber or container 110 for analysis,processing, and/or delivery to another system for analysis and/orprocessing. For example, the sample can be directed from chamber 110 toan automated analyzer 112, such as a bioprofile analyzer available fromNova Biomedical of Waltham, Mass.

The samples that are dispensed from outlet 108 for analysis orprocessing are desirably representative of the materials in bioreactor102 at the time the sample was taken. To reduce the risk ofcontamination, dilution, or alteration of the composition of the samplestaken from sample collection valve 104 and delivered through flow path106, a sanitizing fluid can be delivered through a portion of flow path106 that comes into contact with the samples.

To introduce the sanitizing fluid into flow path 106, a sanitizing fluidinlet valve 114 is provided upstream of sample collection valve 104.Sanitizing fluid inlet valve 114 is operable between a closed positionthat restricts fluid flow through sanitizing fluid inlet valve 114 andan open position that allows fluid flow through sanitizing fluid inletvalve 114. In one embodiment, the sanitizing fluid comprises steam. Insome embodiments, some or all of the valves can be biased closed.

In one embodiment, the sanitizing fluid is any fluid that can sanitize,disinfect, or sterilize the valve. The sanitizing fluid can be a liquid,a gas, or a combination thereof. Sanitizing fluids include steam,ethylene oxide, glutaraldehyde, formaldehyde, formalin, chlorine gas,hypochlorite, bromine, hypobromite, iodine, hypoiodite, brominechloride, chlorine dioxide, ozone, hydrogen peroxide, monochloramine,dichloramine, trichloramine, quatinary ammonium salts, ethanol, 70%ethanol/water, isopropanol, 70% isopropanol/water, peroxyacetic acid,and peracetic acid. In one embodiment, the sanitizing fluid is steam. Inanother embodiment, the sanitizing fluid is ethylene oxide. In anotherembodiment, the sanitizing fluid is glutaraldehyde.

A gas inlet valve 116 can also be provided upstream of sample collectionvalve 104 to deliver a gas through flow path 106. The gas can eliminateand/or reduce the amount of sanitizing fluid remaining within flow path106 after flow path 106 is exposed to the sanitizing fluid. Thesanitizing fluid can clean the path and/or remove any material fromprevious samples in the area contacted by the sanitizing fluid. Gasinlet valve 116 is operable between a closed position that restricts theflow of gas through gas inlet valve 116 and an open position that allowsthe flow of gas through gas inlet valve 116. In one embodiment, the gascomprises compressed air.

To draw a sample from bioreactor 102, a variable volume reservoir 118can be provided downstream of sample collection valve 104. Variablevolume reservoir 118 can be moveable between a first position and asecond position to draw a sample through sample collection valve 104 andinto flow path 106. The sample can be drawn into at least a portion ofvariable volume reservoir 118 along a first portion of flow path 106 anddischarged from variable volume reservoir 118 along a second portion offlow path 106. Variable volume reservoir 118 can comprise a diaphragmpump (as shown in FIG. 1), a syringe pump, or other similar devicecapable of drawing a sample from bioreactor 102.

As shown by dotted lines in FIG. 1, at least a portion of samplingsystem 100 can comprise a unitary structure 125. Thus, for example,unitary structure 125 can comprise sample collection valve 104,sanitizing fluid inlet valve 114, gas inlet valve 116, outlet valve 108,and at least a portion of the fluid flow path. Preferably, the entireflow path between the sanitizing fluid inlet valve 114 and the outletvalve 108 is internal to the unitary structure 125.

FIGS. 2A-2D are schematic representations of the operation of samplingsystem 100. As described in more detail below, sampling system 100 canbe inserted into bioreactor 102 and can operate to sanitize or sterilizea flow path from the point of insertion with bioreactor 102 through theclosed pathway of flow path 106. By being able to sanitize or sterilizethe entire path downstream of the insertion point of sampling system 100into bioreactor 102, the possibility of contaminating bioreactor 102and/or the samples captured from bioreactor 102 is reduced.

FIG. 2A illustrates a sanitizing procedure in which a sanitizing fluid120 (e.g., steam) is directed into flow path 106 through an opensanitizing fluid inlet valve 114. As shown in FIG. 2A, sanitizing fluid120 is directed along flow path 106, including along the portions offlow path 106 that are in contact with samples that are drawn frombioreactor 102 and dispensed from flow path 106. For example, sanitizingfluid 120 is directed along flow path 106 past sample collection valve104, through variable volume reservoir 118, and out outlet valve 108. Assanitizing fluid 120 comes into contact with the internal surfaces thatdefine flow path 106, those surfaces are sanitized or sterilized.

Referring now to FIG. 2B, sanitizing fluid inlet valve 114 is closed andgas inlet valve 116 is opened to allow a gas 122 (e.g., air) to enterflow path 106. As shown in FIG. 2B, gas 122 can also be directed alongflow path 106, including along the portions of flow path 106 thatsanitizing fluid 120 contacts. In this manner, any remaining sanitizingfluid 120 can be purged from flow path 106. If desired, a filter 124(e.g., a sterile air filter) can be provided upstream of gas inlet valve116 to ensure that the gas 122 that enters flow path 106 issubstantially free of impurities and/or contaminants.

FIG. 2C illustrates the operation of variable volume reservoir 118 todraw a sample 126 from bioreactor 102 through open sample collectionvalve 104. As shown in FIG. 2C, variable volume reservoir 118 comprisesa diaphragm pump that moves from a first volume to a second, largervolume as illustrated by arrow 128. The enlargement of the volume ofvariable volume reservoir 118 draws a sample through open samplecollection valve 104 and into flow path 106. Variable volume reservoir118 has an inlet 130 and an outlet 132. After sample 126 is drawn intovariable volume reservoir 118, the diaphragm pump moves from the second,larger volume back to a smaller volume as illustrated by arrow 134 inFIG. 2D. The reduction of the volume of variable volume reservoir 118discharges sample 126 through outlet 132 of variable volume reservoir118. Sample 126 is then discharged through outlet valve 108 to becaptured for analysis and/or further processing.

Referring again to FIG. 1, as sample 126 is discharged through outletvalve 108, it can be delivered to chamber 110. To facilitate delivery ofsample 126 to chamber 110, a control valve 136 can be provideddownstream of outlet valve 108. Control valve 136 can be configured toprovide a back pressure to cause sample 126 to be directed into chamber110 and to provide a desired back pressure along flow path 106 tofacilitate the sanitizing process (e.g., FIG. 2A) and the purgingprocess (e.g., FIG. 2B). Control valve 136 can be configured to open toallow the discharge of waste. The discharged waste can include, forexample, sanitizing fluid and purging gas that has traveled along theflow path 106 to sanitize and purge excess sample materials from flowpath 106.

FIGS. 3A-3F illustrate an exemplary operation of a variable volumereservoir 118. FIG. 3A illustrates variable volume reservoir 118 in afirst configuration with a very small volume (e.g. approximately zerovolume). FIG. 3B illustrates a sample being drawn into variable volumereservoir 118 through inlet 130, thereby moving a diaphragm 140 ofvariable volume reservoir 118 in the direction of arrow 138. Diaphragm140 can continue to move in the direction of arrow 138 and expand thevolume of variable volume reservoir 118 until variable volume reservoir118 reaches a second configuration with a larger volume as shown in FIG.3C. As shown in FIGS. 3D, 3E, and 3F diaphragm 140 can then move fromthe second configuration to the first configuration, causing the samplecontained within variable volume reservoir 118 to be discharged throughoutlet 132.

As shown in FIGS. 3A-3F, the variable volume reservoir 118 can comprisea housing with a first area (e.g., the lower hemispherical portion ofvariable volume reservoir 118 in FIGS. 3A-3F) and a second area thatgenerally opposes the first area (e.g., the upper hemispherical portionof variable volume reservoir 118 in FIGS. 3A-3F). Diaphragm 140 can beconfigured to move between a first position (FIG. 3C) in which anon-sample contacting surface of diaphragm 140 lays generally flush onthe first area of variable volume reservoir 118 so that the volumeavailable for receiving a sample in variable volume reservoir isgenerally maximized and a second position (FIG. 3F) where the diaphragm140 folds in on itself so that a sample contacting surface of diaphragm140 lays generally flush on the second area of variable volume reservoir118. In some embodiments, the surfaces of the diaphragms are notgenerally flush; instead, they are partially deflected, such as shown inFIG. 3B. The amount of deflection can be dependent upon the size of thesample desired.

Thus, as diaphragm 140 moves from the first position (FIG. 3C) to thesecond position (FIG. 3F) to dispense the sample from variable volumereservoir 118, diaphragm 140 folds in on itself to substantially expelfrom variable volume reservoir 118 the entire volume of the sample thatwas received within variable volume reservoir when diaphragm 140 was inthe first position. When diaphragm 140 is in the second position, notonly does diaphragm 140 substantially expel the entire volume of thesample previously contained there, but it also generally forms a barrierto entry into variable volume reservoir 118, thereby at least generallyrestricting entry into variable volume reservoir 118 of any fluid,including the sample that was previously contained therein.

As shown in FIGS. 3A-3F, in some embodiments, diaphragm 140 can comprisea flexible diaphragm in a housing that moves between the first andsecond positions as described herein. The housing can be generallyspherical or it can take other shapes, such as elliptical, pyramidal,top-hat shaped, etc.

Pressure on either side of diaphragm 140 can cause operation of variablevolume reservoir in the manners described herein. For example, pressurefrom the sample source in connection with the delivery of sample 126from bioreactor 102 through open sample collection valve 104 (seeFIG. 1) can actuate variable volume reservoir 118 to cause movement ofdiaphragm into the first position to receive the sample. Alternatively,if the bioreactor is under sub-atmospheric pressure, a negative pressure(i.e., vacuum) can be applied to the “back side” of the diaphragm, andthen switched to a positive pressure for expelling the sample.

Similarly, the sample can be expelled from variable volume reservoir 118by providing pressure on the non-sample contacting surface (i.e., the“back” side) of diaphragm 140. Such pressure can be provided on the backside of diaphragm 140 by delivering, for example, any fluid towards thatsurface as indicated, for example, by the origination of the arrow inFIGS. 3D and 3E.

Thus, in some embodiments, diaphragm 140 can comprise a flexible,inverting diaphragm that can advantageously provide a system that iscapable of performing a pumping action with relatively low amounts ofturbulence being introduced to the system. In addition, such a diaphragmis capable of actuation using relatively low pressures, including arelatively low sample inlet pressure to move the diaphragm from thesecond position (FIG. 3A, 3F) to the first position (FIG. 3C).

In some embodiments, the flexible material can have a low gaspermeability to ensure the fluid used to expel the sample (e.g., air ornitrogen) doesn't permeate into the sample, thus changing theproperties. EPDM can be used for the flexible material. Alternatively,other materials such as Kalrez®, Viton®, polyethylene, polyurethane, andpolypropylene can also be used. The material should be able to withstandthe sanitization conditions—e.g., steam.

FIGS. 4A-4D illustrate another embodiment of a sampling system 200.Sampling system 200 is generally similar to sampling system 100 and likeelements are identified by similar reference numbers. The maindifferences between sampling system 100 and 200 are illustrated in thevarious figures and described in the related descriptions of thosesystems as included herein.

Sampling system 200 can include a sample collection valve 204, an outletvalve 208, a sanitizing fluid inlet valve 214, and a gas inlet valve216. One or more of these valves can be configured to have a valve stem221 and a sealing member 223. Although FIG. 4A illustrates each of thesevalves as having a valve stem 221 and a sealing member 223, it should beunderstood that the type of valve can vary. The valve stems can beactuated by springs or air, and preferably by a combination of spring-and air-actuation.

FIG. 4A illustrates a sanitizing or sterilizing process. During theprocess shown in FIG. 4A, sample collection valve 204, outlet valve 208,and gas inlet valve 216 are closed with sealing members 223 moved intoengagement with the respective openings associated with those valvesinto flow path 206. Thus, for example, the sealing member 223 of samplecollection valve 204 is engaged with an opening between flow path 206and bioreactor 202 to restrict the passage of material in bioreactor 202from entering flow path 206. At least a portion of the valve stem 221associated with the sample collection valve 204 extends into flow path206, but does not entirely block flow path 206. In this manner,sanitizing fluid can pass across a portion of the sample collectionvalve 204 (and other valves in a similar manner) to sterilize andsanitize the portions of the valve that is in flow path 206. Thus, asshown in FIG. 4A, sanitizing fluid is directed through flow path 206across the closed gas inlet valve 216, across the closed samplecollection valve 204, through the variable volume reservoir 218, acrossthe closed outlet valve 208, and out an open control valve 236.Contaminants and other materials caught up in the sanitizing fluid canalso exit control valve 236.

Referring to FIG. 4B, the sanitizing fluid inlet valve 214 can be closedand gas inlet valve 216 can be opened to deliver a purging gas (e.g.,air) through flow path 206 to remove and/or reduce the presence ofsanitizing fluid within flow path 206. The gas is directed through flowpath 206 across the closed gas inlet valve 216, across the closed samplecollection valve 204, through the variable volume reservoir 218, acrossthe closed outlet valve 208, and out the open control valve 236.

Once the gas purges the remaining sanitizing fluid from flow path 206,both the sanitizing fluid inlet valve 214 and the gas inlet valve 216can close to allow a sample to be drawn into flow path 206. As shown inFIG. 4C, variable volume reservoir 218 draws a sample through the opensample collection valve 204 and into the volume of variable volumereservoir 218. Variable volume reservoir 218 then directs the drawnsample further downstream along flow path 206 towards outlet valve 208as shown in FIG. 4D. Outlet valve 208 can open to allow the sample to bedischarged from flow path 206.

FIGS. 5A - 5C illustrate enlarged views of exemplary valves that can beused with the systems disclosed in FIGS. 4A - 4C. For example, FIGS. 5Aand 5B illustrate a three-way bypass flow valve that can move between anopen configuration (FIG. 5B) and a closed configuration (FIG. 5A). InFIG. 5A, valve stem 221 is shown extending into flow path 206 withsealing member 223 closing a port 231 (e.g., a gas inlet port, a samplecollection inlet port, a sample collection outlet port) into flow path206. In the closed configuration, fluid can flow past valve stem 221 asshown by arrow 225. One or more sealing rings 233 (e.g., 0-rings) can atleast partially surround valve stem 221 to restrict the flow of fluidout of flow path 206 in the area of valve stem 221. In addition, a weephole 235 can be provided to further remove any moisture of other fluidsthat may move past sealing rings 233.

A spring 237 can be provided to bias valve stem 221 towards the closedconfiguration (FIG. 5A) and to ensure that sealing member 223 seatsitself properly with port 231. An air inlet 239 can be provided adjacentvalve stem 221 to move valve stem 221 from the closed configuration(FIG. 5A) to the open configuration (FIG. 5B). Compressed air or otherfluids can be directed through air inlet 239, causing valve stem 221 tomove downward as shown in FIG. 5B. As valve stem 221 moves downward,sealing member 223 moves out of engagement with port 231, allowing fluidto pass through port 231 and enter flow path 206 as shown by arrow 241.

FIG. 5C illustrates a two-way valve that is moveable between a closedconfiguration (not shown) and an open configuration (FIG. 5C). As shownin FIG. 5C, a valve stem 221 with a sealing member 223 can move into anopen configuration in the same manner as that shown in FIG. 5B. Such avalve can be used, for example, with a port 231 that is configured to beopened and closed to allow fluid to flow into the pathway, such as asanitizing fluid inlet port or a waste outlet port.

FIG. 6 illustrates a cross-sectional view of a portion of anotherexemplary sampling system 200, shown with an angled fluid path 206between the sanitizing fluid inlet valve 214 and the outlet valve 208.Sample collection valve 204 extends from a main body 235 of samplingsystem 200 to facilitate coupling of sample collection valve 204 withbioreactor 202 (not shown in FIG. 6).

FIGS. 7 and 8 illustrate views of portions of another exemplary samplingsystem 200, also having an angled fluid path 206 between the sanitizingfluid inlet valve 214 and the outlet valve 208. As shown in the enlargedpartial cross-sectional view of FIG. 8, when sample collection valve 204is in a closed position (e.g., with a sealing member 223 extending intoan opening between the bioreactor and flow path 206), sanitizing fluidcan flow around the end of valve stem 221. Thus, for example, as shownby arrows 255, sanitizing fluid can pass around a portion of samplecollection valve 204, thereby improving sanitization or sterilization ofthe area adjacent the opening extending into the bioreactor.

Moreover, by forming sample collection valve with a sealing member 223that tapers from valve stem 221, the area of contact between sealingmember 223 and the opening can be reduced. To provide improved sealingcharacteristics, in some embodiments, the tip of the valve stem canextend at an angle of greater than 50 degrees from the body of the valvestem and, more preferably at an angle of greater than 70 degrees and,even more preferably at an angle of about 80 degrees.

In some embodiments, sealing member 223 (FIG. 8) can be formed of apolymeric material that is softer than the material of the seat, intowhich sealing member 223 extends. In some embodiments, the seat can beformed of a more rigid polymer material. For example, the seat can bemade of most teflons (PTFE, PFA, ETFE, etc.) and the seat can be made ofa high performance, high temperature, harder thermoplastic. (PEEK, PEI,PPSU, PSU, etc.). For the stem, creep resistant PFA is preferred andPEEK is preferred for the rest of the body. PEEK and teflons arepreferable materials due to their relatively chemically inert behavior.

In this manner, sealing member 223 can extrude into the seat to form atighter seal. In addition, as shown in FIG. 7, sealing member 223 canhave a steeper cone shape than the hollow cone seat, thereby allowingsealing member 223 to extrude into the seat to form a positive seal. Thedesign allows for a variable sealing area, causing the stem to deformuntil the stress on the materials at the seal is within the elasticmodulus of the valve stem, allowing a good seal even with relativelywide tolerances on the angles of the seat and stem. This configurationcan have several advantages. For example, because the seat can be formedas a cone-shaped hole as shown in FIG. 7, the opening can be very small,allowing it to be easily incorporated into a small conduit and amenableto sterilization in the manners described herein (e.g., by steam).Moreover, when one or both of the sealing member and seat are formed ofpolymers, the heat up and cool down times associated with those partscan be faster than the times associated with other materials, such assteel or other metals.

In some embodiments, the sealing member and valve stem can be formed ofthe same polymeric material, which can further improve operation byreducing complexities of manufacturing and permitting the sealing memberand valve stem component to be more compact.

FIG. 9 illustrates another embodiment of sampling system 200, with avariable volume reservoir 218 integrally formed with the sampling systemstructure. Variable volume 218 comprises a diaphragm pump connected flowpath 206 to draw samples from the bioreactor (not shown) to whichsampling system 200 is coupled.

As described above with respect to FIG. 1, a control valve 136 can beprovided downstream of outlet valve 108. Control valve 136 can beconfigured to provide a desired back pressure along flow path 106 tofacilitate the sanitizing process (e.g., FIG. 2A), the purging process(e.g., FIG. 2B), and/or the sample collection process (e.g., FIG. 2D).For example, during the sanitizing process, it is desirable to keep thesanitizing fluid at a desired temperature for a desired length of time(e.g., if steam is the sanitizing fluid it can be desirable to maintainthe steam at about 121° C.). By providing back pressure via the controlvalve, the control valve can help direct the sample and the temperaturewithin the flow path during the sanitizing process can be more easilymaintained.

FIGS. 10-12 illustrate an embodiment of a control valve 136 thatcomprises a diaphragm valve. As shown in FIGS. 11 and 12, control valve136 can comprise a diaphragm 191 positioned between two wall members193, 195 to restrict and/or allow flow through the control valve. Forexample, first wall member 193 can comprise an inlet 196 and an outlet197. Movement of diaphragm 191 towards first wall member 193 restrictspassage of fluid through inlet 196 and outlet 197. To provide formovement of diaphragm 191, a control air inlet 199 can be provided onthe opposing second wall member 195. An increase in air pressure atcontrol air inlet 199 causes diaphragm 191 to move towards first wallmember 193, while a decrease in air pressure at control air inlet 199causes diaphragm 191 to move away from first wall member 193. In thismanner, back pressure can be adjusted adjacent the outlet valve of thesampling system as needed or desired.

Referring again to FIG. 10, a holding coil 189 can be provided tocontain a sample during a sample collection processing. Holding coil 189can provide a volume into which a sample can be drawn. In operation, thesample is pumped or drawn into holding coil 189 and then drawn into thechamber from holding coil 189. This can allow larger samples to be drawnand, if the sample drawn is larger than the sample delivered intochamber 110, ensure that the sample delivered into chamber 110 is from acentral region of the drawn sample. By capturing a central portion ofthe sample, the likelihood of that sample being contaminated within theflow path of the sampling system can be further reduced.

FIGS. 13A-13D illustrate various views of an integral sampling system300. As in other embodiments, sampling system 300 includes a samplecollection valve 304, an outlet valve 308, a sanitizing fluid inletvalve 314, a gas inlet valve 316, and a flow path 306 extending alongthese valves. A variable volume reservoir 318 can comprise a diaphragmpump that is configured to draw fluid from a bioreactor through an opensample collection valve 304. Sampling system 300 can be formed of anintegral structure that can be coupled to a bioreactor to drawn samplestherefrom.

As discussed above, the variable volume reservoirs can include adiaphragm pump or other similar structures. FIG. 14 illustrates asampling apparatus 400 that comprises a variable volume reservoir 418.Sampling apparatus 400 can generally function similar to other samplingapparatuses described herein. However, instead of the diaphragm pumpsillustrated in the other embodiments, variable volume reservoir 418 is asyringe-type pump. Thus, by operating the syringe-type pump to increasea volume in variable volume reservoir 418, the sample is drawn throughopen sample collection valve 404, into flow path 406, and into thereservoir of the syringe-type pump. As the volume in the syringe-typepump is decreased, the sample is discharged from the reservoir of thevariable volume reservoir 418 and out the outlet valve 408.

EXAMPLE Comparison of Sampling System Valve to Conventional ManualSampling

An automated aseptic sampling (AAS) system similar to that describedabove with respect to FIG. 1 was installed on a bioreactor to drawsamples and deliver them to an analyzer without contamination and gasexchange, while maintaining sterility of the bioreactor. Automaticsamples taken using the AAS system were compared to conventional manualsampling techniques. Manual samples were taken from the chamber andintroduced to a Nova® Biomedical Bioprofile® FLEX autosampler (Waltham,Mass.) using a syringe. The samples were then analyzed to compareresults.

For the first comparison test, the AAS system was attached to a 30 L NewBrunswick bioreactor (Edison, N.J.) containing NS0 culture grown inmedia. As the AAS was drawing sample through an independent port, manualsamples were drawn to provide a sample pair. AAS and manual samples wereboth introduced to the FLEX autosampler for analysis of pH and carbondioxide (measuring cell activity). Sample error was defined as thedifference between the AAS system and manual sampling for a singlesample pair. Errors from the sample pairs were averaged to determinevariability. The results are shown in Tables 1 and 2 and demonstratethat the AAS is as accurate as manual sampling.

TABLE 1 Analysis of pH Results Sample pH pH ID AAS Manual Difference 17.196 7.196 0 2 7.183 7.175 0.008 3 7.198 7.181 0.017 4 7.177 7.1630.014 5 7.182 7.166 0.016 Average Difference 0.011

TABLE 2 Analysis of Carbon Dioxide Results Sample pCO₂ (mmHg) pCO₂ IDAAS Manual Error (%) 1 58.3 56.9 2.5% 2 60.9 61.3 0.7% 3 57.1 57.3 0.3%4 59.1 61.1 3.3% 5 57.6 60.9 5.4% Average Difference 2.4%For the second comparison test, an aliquot of cells was added to astainless steel cylindrical vessel. The vessel was inverted severaltimes to mix. Automatic samples were taken by the AAS system attached tothe bottom of the vessel. Manual samples were removed via pipettethrough the top of the vessel. Automatic and manual samples wereintroduced to the FLEX autosampler, and sample pairs were analyzed forcomparison. Results in Table 3 demonstrate accurate sampling using theAAS valve of the invention.

TABLE 3 Cell Count Testing Results Total Density Viable Density Sample(cells/mL) Total Density (cells/mL) Viable Density Viability (%)Viability ID AAS Manual Error AAS Manual Error AAS Manual Error 1 61.3170.26 −12.7% 57.69 65.75 −12.3% 94.1 93.6 0.5% 2 31.43 33.15 −5.2% 29.5831.07 −4.8% 94.1 93.7 0.4% 3 125.38 135.66 −7.6% 119.89 128.86 −7.0%95.6 95 0.6% 4 136.08 133.63 1.8% 124.73 123.13 1.3% 91.7 92.1 −0.4% 5125.35 141.11 −11.2% 116.34 130.05 −10.5% 92.8 92.2 0.7% 6 71.15 72.52−1.9% 64.6 66.17 −2.4% 90.8 91.2 −0.4% 7 36.14 36.93 −2.1% 32.23 33.32−3.3% 89.2 90.2 −1.1% 8 126.45 141.59 −10.7% 116.61 130.89 −10.9% 92.292.4 −0.2% 9 135.59 137.99 −1.7% 126.65 127.56 −0.7% 93.4 92.4 1.1% 10147.26 135.35 8.8% 136.28 125.44 8.6% 92.5 92.7 −0.2% 11 80.75 72.5411.3% 69.36 66.31 4.6% 85.9 91.4 −6.0% 12 153.91 135.63 13.5% 133.44124.65 7.1% 86.7 91.9 −5.7% 13 137.91 137.47 0.3% 127.28 125.68 1.3%92.3 91.4 1.0% 14 62.96 72.34 −13.0% 57.38 64.81 −11.5% 91.1 89.6 1.7%15 38.68 40.28 −4.0% 34.54 35.06 −1.5% 89.3 87 2.6% 16 139.07 143.25−2.9% 128.15 130.79 −2.0% 92.1 91.3 0.9% 17 74.44 73.56 1.2% 67.49 66.721.2% 90.7 90.7 0.0% 18 32.98 36.93 −10.7% 30.21 32.91 −8.2% 91.6 89.12.8% 19 147.19 136.29 8.0% 138.86 126.41 9.8% 94.3 92.7 1.7% 20 131.98135.08 −2.3% 122.34 124.3 −1.6% 92.7 92 0.8% Average 6.5% 5.5% 1.4%Error *Note: NS0 cells grown in fed-batch culture were removed frombioreactor just prior to addition into sampling vessel.

The prototype AAS system of the invention was further tested todemonstrate long-term operation and removal of representative bioreactorsamples without bioreactor contamination. The AAS system was mounted ona 30-L bioreactor and tested, using the test schematic noted above,which is similar to that shown in FIG. 1. The AAS valve was cycled every30 minutes for 3 weeks, for a total of more than 1200 cycles. Cyclesconsisted of heating the valve to more than 121° C. (measured at theoutlet) for 20 minutes and then cooling it to about 50° C. beforedispensing a 20-mL sample. Five days before the end of the test, thevalve was intentionally contaminated five times with E. coli (once onone day and twice a day for two days) with no resulting contamination ofthe bioreactor. These tests demonstrate successful operation of the AASsystem.

Additional tests of the AAS/Flex autosampler were performed at the 30-Lscale. At a scheduled time point, the AAS system transferred a samplefrom the bioreactor to the FLEX and commanded the FLEX to take thedelivered sample and analyze it. It then cleaned and sanitized itself inpreparation for the next sample. A manual sample was taken within about15 minutes of the automated sample. Cell density and viabilitymeasurements from the AAS/Flex autosampler were compared to measurementsfrom manual sampling, to demonstrate that the AAS/Flex autos amplerresults are in agreement with results obtained from manual samples.Comparison of results in Table 4 shows accurate sampling using the AASvalve of the invention.

TABLE 4 Cell Count Performance of the AAS/Flex Autosampler @ 30 L ScaleViable Cell Density % % (1E6 cells/mL) Differ- Viability (%) Differ- DayAAS Manual ence AAS Manual ence 0.00 0.70 n/a n/a 96.90 n/a n/a 0.370.87 0.80 8.06 98.70 98.90 0.20 0.82 1.02 0.88 13.78 99.00 99.00 0.001.84 2.05 1.67 18.60 99.40 99.20 0.20 3.06 3.55 3.54 0.28 98.10 98.400.30 3.40 4.58 4.38 4.47 98.40 98.20 0.20 3.83 5.19 4.55 12.39 98.8098.80 0.00 4.10 5.94 5.93 0.29 98.70 98.70 0.00 4.41 6.65 6.20 6.7998.90 98.90 0.00 4.80 7.15 7.51 5.14 99.30 99.10 0.20 5.42 9.18 8.645.91 99.10 98.80 0.30 5.80 10.05 9.93 1.13 99.30 99.10 0.20 6.08 11.7011.32 3.26 99.20 99.20 0.00 6.39 11.78 11.20 4.92 99.20 99.20 0.00 6.8012.39 12.13 2.10 98.80 98.90 0.10 7.10 13.13 13.71 4.42 98.30 98.20 0.107.38 14.30 12.34 13.72 98.00 98.10 0.10 7.86 14.59 14.63 0.29 98.2098.00 0.20 8.93 15.06 14.72 2.27 96.80 96.60 0.20 9.78 14.93 15.10 1.1195.20 95.30 0.10 10.11 14.86 15.28 2.79 94.90 94.10 0.80 10.40 14.6915.40 4.83 94.70 93.90 0.80 11.91 14.05 14.24 1.30 92.70 93.00 0.3012.70 13.16 12.84 2.44 91.00 91.50 0.50 13.77 12.05 12.21 1.29 87.4089.50 2.10 Average % 5.07% 0.29% Difference

Results in Table 5 show pH measurements of samples taken using theAAS/Flex autosampler compared to measurements from manual sampling.Table further demonstrates accurate sampling using the AAS valve of theinvention.

TABLE 5 pH Performance of the AAS/Flex Autosampler @ 30 L Scale pH DayAAS Manual Difference 0.37 7.232 7.221 0.011 0.82 7.221 7.241 0.02 1.847.171 7.227 0.056 3.06 6.93 6.943 0.013 3.4 6.936 6.952 0.016 3.83 6.9276.95 0.023 4.1 6.922 6.943 0.021 4.41 6.927 6.945 0.018 4.8 6.918 6.9290.011 5.42 6.917 6.951 0.034 5.8 6.906 6.924 0.018 6.08 6.894 6.9420.048 6.39 6.903 6.943 0.04 6.8 6.915 6.932 0.017 7.1 6.912 6.955 0.0437.38 6.938 6.995 0.057 7.86 6.971 7.003 0.032 8.93 7.01 7.056 0.046 9.787.018 7.066 0.048 10.11 7.035 7.088 0.053 10.40 7.042 7.102 0.06 11.917.116 7.161 0.045 13.77 7.077 7.119 0.042 Average Unit 0.034 ofDifference

Results in Table 6 show pCO₂ measurements of samples taken using theAAS/Flex autosampler compared to measurements from manual sampling.Table 6 further demonstrates accurate sampling using the AAS valve ofthe invention.

TABLE 6 pCO₂ Performance of the AAS/Flex Autosampler @ 30 L Scale pH DayAAS Manual % Difference 0.37 69.6 70.8 1.72 0.82 64.7 60.4 6.65 1.8443.0 35.6 17.21 3.06 27.0 25.5 5.56 3.40 30.7 29.1 5.21 3.83 36.9 31.714.09 4.10 36.9 38.1 3.25 4.41 39.6 37.8 4.55 4.80 42.2 40.8 3.32 5.4242.6 38.9 8.69 5.80 42.5 41.6 2.12 6.08 42.9 41.2 3.96 6.39 47.5 42.310.95 6.80 47.5 45.9 3.37 7.10 48.1 47.0 2.29 7.38 47.3 39.9 15.64 7.8646.8 45.8 2.14 8.93 48.7 42.2 13.35 9.78 49.0 46.5 5.10 10.11 49.6 46.85.65 10.40 51.4 48.4 5.84 11.91 58.2 51.1 12.20 12.70 76.7 73.7 3.9113.77 95.9 88.3 7.92 Average % 6.86 Difference

Results in Table 7 show osmolarity measurements of samples taken usingthe AAS/Flex autosampler compared to measurements from manual sampling.Table 7 further demonstrates accurate sampling using the AAS valve ofthe invention.

TABLE 7 Osmolarity Measurement of the AAS/Flex Autosampler @ 30 L ScalepH Day AAS Manual % Difference 0.37 351 329 6.27 0.82 349 325 6.88 1.84342 306 10.53 3.06 348 352 1.15 3.4 354 327 7.63 3.83 362 341 5.80 4.1366 360 1.64 4.41 370 343 7.30 4.8 375 374 0.27 5.8 379 375 1.06 6.08381 380 0.26 6.39 380 356 6.32 6.8 383 376 1.83 7.1 382 375 1.83 7.38380 350 7.89 7.86 385 378 1.82 8.93 377 344 8.75 9.78 373 377 1.07 10.11375 371 1.07 10.40 376 373 0.80 11.91 380 383 0.79 13.77 388 396 2.06Average % 3.77 Difference

Exemplary Applications of Various Systems and Methods Disclosed Herein

As described herein, optimal production in bioreactors requires regularsampling for off-line analysis to ensure the process remains within thedesired operating space for maximum product production. The automatedvalve disclosed herein, the Automated, Aseptic Sampling (AAS) system canprovide rapid, closed-cycle sampling of the bioreactor, steam-in-place(SIP) sterilization between samples, and direct sample delivery to ananalyzer. The AAS not only automates and facilitates the samplingprocess, but can also provide greater reproducibility when compared tomanual sampling and has the additional benefits of safety andreliability. The automated sample scheduling and communication with theanalytical devices enhances the ability to integrate with processcontrol strategies.

The AAS was installed on 30-L and 130-L bioreactors. Samples werecollected using the sampling system and analyzed using a NOVA Flexanalyzer (Waltham, Mass.). Outputs from the analyzer included viablecell concentration, cell viability, glucose, pH, partial pressure ofcarbon dioxide, and osmolality. The AAS system demonstrated the abilityto take 3-20× more samples compared to the conventional manual methodstypically used, over long periods of time, without affecting theintegrity of the bioreactor process. The system performed moreconsistently and reliably than when samples were taken manually in thedevelopment area and showed improved reproducibility.

Some features of the design of the AAS and its sample cycle (as testedin this example) are provided below.

Design

-   -   Compact/self-contained with on-board, closed-cycle, sample pump    -   Current Good Manufacturing Practice (cGMP) compliant    -   OPC communication capable for integration with variety of        analyzers and devices    -   Unique valve design    -   Scheduler with operator-specified sampling intervals        Sample Cycle (<45 minutes)    -   SIP for sterilization    -   Cool down followed by condensate purge    -   Sample draw    -   Sample dispense to sample-handling device or directly to        analytical instrument

In tests with the AAS system, more than 500 samples were taken in afour-week-long test; while more than 150 samples were taken in three,two-week-long tests. In all instances, the system performed moreconsistently and reliably than when samples were taken manually, and theAAS showed improved reproducibility. No system contamination occurredduring these tests. During the one testing period, 99 samples were takenwithout any impact on the sterility of the bioreactor.

The improved performance of the AAS system over manual sampling makes itdesirable for use in bioreactors. The AAS demonstrates reliablecontamination-free sampling with greater sample consistency andreproduction when compared to manual samples. This scale-independent,low-cost sampling system, which can be manufactured from cGMP-compliantmaterials, is capable of frequent sampling to enable more intensiveprocess-control schemes. Savings in labor and processoptimization/efficiency can be achieved. Moreover, the highly efficientAAS also has use in disposable systems and downstream applications.

The automated sampling systems described herein can advantageously allowfor more frequent collection of data, reduce sampling variation andhuman error associated with the capturing of samples, and reduce costsby reducing labor requirements associated with manual sampling.

It should be understood that the various steps of the disclosed methodsand the various components of the disclosed apparatuses are exemplaryand the particular order of steps and arrangement of components can bevaried without departing from the scope of the invention. For example,FIGS. 26-29 illustrate additional embodiments with various components ofthe apparatus rearranged, resulting in variations in the order and/ormanner in which the steps of the respective methods are performed. Theseadditional embodiments are merely examples of some of the manners inwhich the steps and/or components of the disclosed embodiments can berearranged without departing from the scope of the invention. Otherrearrangement of steps and/or components are contemplated.

FIG. 23 illustrates an exemplary embodiment of a sampling device 500 inwhich the introduction of a sanitizing fluid (e.g., steam) and a purgefluid (e.g., air) into a fluid flow path of the apparatus occurs througha common valve device 501 (e.g., a three-way valve device). In thismanner, the sanitizing fluid (e.g., steam) and purge fluid (e.g., air)can enter into the fluid flow path 506 at a common location rather thanat separate locations as shown, for example, in FIG. 1.

In operation, for example, a sanitizing fluid (e.g., steam) can bedelivered into fluid flow path 506 via a three-way valve 501 to cleanthe path and/or remove any material from previous samples in the areacontacted by the sanitizing fluid. After the sanitizing step, a purgefluid (e.g., air) can be delivered to the fluid flow path 506 via thesame valve 501. Because valve 501 is upstream of sample collection valve504 (which is, in turn, coupled to the bioreactor 502), the air caneliminate and/or reduce the amount of sanitizing fluid remaining withinfluid flow path 506 after fluid flow path 506 is exposed to thesanitizing fluid. Thus, three-way valve 501 is operable between a firstposition that restricts the flow of air (or other purging fluid) butpermits sanitizing fluid to pass through, a second position that permitsthe flow of air (or other purging fluid) and restricts the passage ofsanitizing fluid, and a third position that restricts the flow of bothair (or other purging fluid) and the sanitizing fluid.

The remaining operation of the device illustrated in FIG. 23 can begenerally similar to that described elsewhere herein. For example, avariable volume reservoir 518 can draw a sample from bioreactor 502through an open sample collection valve 504. The variable volumereservoir 518 can comprise, for example, a diaphragm pump as describedelsewhere herein. After the sample is drawn into variable volumereservoir 518, it can be discharged through outlet valve 508 to becaptured for analysis and/or further processing by an analyzer 512. Tofacilitate delivery of the sample to the analyzer 512, a control valve536 can be provided downstream of outlet valve 508. As describedelsewhere herein, a control valve 536 can be provided to open and closeto allow and restrict the discharge of fluids to a waste collectionarea. The discharged waste can include, for example, sanitizing fluidand purging gas that has traveled along fluid flow path 506 to sanitizeand purge excess sample materials from fluid flow path 506.

FIG. 24 illustrates another exemplary embodiment of a sampling device600. In this embodiment, the sanitizing fluid (e.g., steam) and purgefluid (e.g., air) are also configured to be delivered into a fluid flowpath 606 at a common location (e.g., via a three-way valve 601) asdescribed in FIG. 23. FIG. 24, however, illustrates an alternativeembodiment in which the components and steps downstream from the sampleinlet valve are arranged differently from other embodiments disclosedherein.

As shown in FIG. 24, a sample collection valve 604 is coupled to abioreactor 602 and a variable volume reservoir 618 is provideddownstream of the sample collection valve 604. A three-way valve 615 isprovided downstream from variable volume reservoir 618, with valve 615being configured to permit delivery of a sample and/or other materialsin fluid flow path 606 through valve 615 to an analyzer 612 or,alternatively, to a waste collection area. Accordingly, when capturing asample, the system draws the sample into variable volume reservoir 618and valve 615 moves to a first configuration which permits the deliveryof the sample through valve 615 to analyzer 612. After samplecollection, valve 615 moves to a second configuration which restrictsfluid flow to analyzer 612 and permits fluids in the flow path 606(e.g., sanitizing fluid and purging gas) to be directed to a wastecollection area.

FIG. 25 illustrates another exemplary sampling apparatus 700. Apparatus700 comprises a three-way valve 701 and a bioreactor 702 coupled to asample collection valve 704. Unlike other embodiments disclosed herein,a variable volume reservoir 718 can draw a sample “upstream” throughsample collection valve 704. Once the sample is drawn, the samplecollection valve 704 can close and the variable volume reservoir 718 candischarge the sample back “downstream” along fluid flow path 706 towardsa three-way valve 715 that is provided downstream from variable volumereservoir 718. Valve 715, can be configured to permit delivery of thesample and/or other materials in the fluid flow path 706 through valve715 to an analyzer 712 or to discharge as waste. Accordingly, whencapturing a sample, the system draws the sample “upstream” into variablevolume reservoir 718 and variable volume reservoir 718 then delivers thesample through valve 715 to analyzer 712. After sample collection anddischarge to analyzer 712, valve 715 can restrict fluid flow to analyzer712 and instead direct fluids in the flow path 706 (e.g., sanitizingfluid and purging gas) through valve 715 to a different path for wastecollection.

FIG. 26 illustrates another exemplary sampling apparatus 800. As inother systems described herein, apparatus 800 comprises a bioreactor 802coupled to a sample collection valve 804. Other components of apparatus800, however, are rearranged to operate somewhat differently from othersystems described herein. For example, although the sanitizing fluidinlet is positioned upstream (e.g., at three-way valve 821) of samplecollection valve 804, a purging fluid inlet is positioned downstream ofsample collection valve 804. In operation, variable volume reservoir 818can draw a sample “downstream” through sample collection valve 804. Oncethe sample is drawn, sample collection valve 804 can close and thevariable volume reservoir 818 can deliver the sample “upstream” alongfluid flow path 806 towards three-way valve 821. Valve 815 can beconfigured to permit delivery of the sample through valve 815 toanalyzer 812. Thus, when capturing a sample, the system draws the sample“downstream” into variable volume reservoir 818 and variable volumereservoir 818 then delivers the sample back “upstream” through valve 815to analyzer 812. After sample collection and delivery to analyzer 812,valve 815 is closed so that sanitizing fluid can be delivered throughvalve 821 and along fluid flow path 806 to sanitize fluid flow path 806.After sanitization, a purge fluid (e.g., air) can be delivered into thefluid flow path 806 (as shown in FIG. 26) and valve 821 can be directedto waste, and control valve 836 can be opened to allow discharge offluids in the flow path 806 (e.g., sanitizing fluid and purging gas) forwaste collection.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

We claim:
 1. A sampling system for collecting a fluid sample from anenclosed container, the sampling system comprising: (a) a sanitizingfluid inlet valve; (b) a gas inlet valve; (c) a sample collection valveoperable between an open position that allows a fluid sample to flowfrom the enclosed container into a fluid flow path and a closed positionthat restricts the flow of fluid from the enclosed container to thefluid flow path; (d) a first outlet valve operable between an openposition and a closed position; and (e) a variable volume reservoircomprising a reservoir inlet, a reservoir outlet, and a reservoir havinga volume that can change from a first volume to a second volume when (c)is in the closed position, with the second volume being smaller than thefirst; wherein the fluid flow path interconnects (a) - (e), wherein when(a) and (b) are oriented to restrict flow of the sanitizing fluid andgas through the respective valves and (d) is in the closed position, (c)can be in the open position to withdraw a fluid sample from the enclosedcontainer into a first portion of the fluid flow path for delivery tothe variable volume reservoir that is located downstream from the firstportion of the fluid flow path, wherein when (a) and (b) are oriented torestrict flow of the sanitizing fluid and gas through the respectivevalves and (c) is in the closed position, the fluid sample can bedischarged from the variable volume reservoir along a second portion ofthe fluid flow path through (d), and wherein when (a) is oriented topermit flow of the sanitizing fluid through the sanitizing fluid inletvalve, (b) is oriented to restrict flow of the gas through the gas inletvalve, and (c) is in the closed position, a sanitizing fluid can beintroduced into the fluid flow path through (a) to sanitize at least thefirst portion of the fluid flow path, wherein the variable volumereservoir comprises a pump that is configured to draw the fluid samplein the first portion of the fluid flow path into the variable volumereservoir through the reservoir inlet and direct the fluid sample out ofthe variable volume reservoir into the second portion of the fluid flowpath through the reservoir outlet.
 2. The sampling system of claim 1,wherein when (a) is in the open position and (b) and (c) are in theclosed position, the sanitizing fluid also sanitizes the variable volumereservoir.
 3. The sampling system of claim 1, wherein (a) is at anupstream portion of the fluid flow path and (d) is at a downstreamportion of the fluid flow path, and the sanitizing fluid can flowthrough the fluid flow path from (a) to (d) to sanitize the fluid flowpath between (a) and (d).
 4. The sampling system of claim 3, wherein(a) - (e) are interconnected along the fluid flow path from the upstreamportion to the downstream portion in the following order: (a), (b), (c),(e), and (d).
 5. The sampling system of claim 1, wherein the sanitizingfluid inlet valve is operable between an open position and a closedposition, the gas inlet valve is operable between an open position and aclosed position, and the sanitizing fluid inlet valve and gas inletvalve are separate valves.
 6. The sampling system of claim 1, whereinthe sanitizing fluid inlet valve and the gas inlet valve are coupled viaa three-way valve, the three way valve being operable between a firstposition that permits the flow of sanitizing fluid through thesanitizing fluid inlet into the fluid flow path, a second position thatpermits the flow of gas through the gas inlet into the fluid flow path,and a third position that restricts the flow of both sanitizing fluidand gas into the fluid flow path.
 7. The sampling system of claim 1,wherein the variable volume reservoir comprises a diaphragm pump.
 8. Thesampling system of claim 1, wherein the variable volume reservoircomprises a flexible diaphragm member and a housing with a first areaand a second area, the flexible diaphragm member being configured tomove between a first position where a first surface of the flexiblediaphragm member contacts the first area of the housing and a secondposition where a second surface of the flexible diaphragm membercontacts the second area of the housing.
 9. The sampling system of claim8, wherein the first surface of the flexible diaphragm member isgenerally opposite the second surface of the flexible diaphragm member.10. The sampling system of claim 8, wherein the housing comprising agenerally spherical housing and the first area comprises a first half ofthe generally spherical housing and the second area comprises a secondhalf of the generally spherical housing, the first and second halves ofthe generally spherical housing being generally opposite one another.11. The sampling system of claim 8, wherein the housing isnon-spherical.
 12. The sampling system of claim 10, wherein the flexiblediaphragm member is configured so that the second surface contacts thesecond half of the generally spherical housing when in the secondposition, the flexible diaphragm member being generally inverted when inthe second position, relative to its orientation when in the firstposition.
 13. The sampling system of claim 1, further comprising asecond outlet valve, the second outlet valve being located downstream ofthe first outlet valve, wherein when (a) is oriented to permit flow ofthe sanitizing fluid through the sanitizing fluid inlet valve, (b) isoriented to restrict flow of the gas through the gas inlet valve, and(c) and (d) are in the closed position, the sanitizing fluid can flowalong the fluid flow path between (a) and the second outlet valve tosanitize portions of the fluid flow path in the vicinity of (c) and (d).14. The sampling system of claim 13, wherein the second outlet valvecomprises a variable back-pressure regulator.
 15. The sampling system ofclaim 13, wherein the second outlet valve comprises athermostatically-controlled valve.
 16. The sampling system of claim 13,wherein when (a) is oriented to restrict flow of the sanitizing fluidthrough the sanitizing fluid inlet valve and (c) is in the closedposition, and (b) is oriented to permit flow of the gas through the gasinlet valve, and (d) and the second outlet valve are in the openposition, gas can be introduced into the fluid flow path through (b) topurge the sanitizing fluid from at least the first and second portionsof the fluid flow path.
 17. The sampling system of claim 1, wherein thesample collection valve comprises a valve stem with a tapered sealingmember, and a portion of the valve stem extends into the fluid flow pathwhen the sample collection valve is in the closed position so thatsanitizing fluid introduced into the fluid flow path by the sanitizingfluid inlet valve will flow past the portion of the valve stem thatextends into the fluid flow path.
 18. A method of collecting a fluidsample from an enclosed container, the method comprising: opening asanitizing fluid inlet valve and directing sanitizing fluid downstreamthrough a fluid flow path past a sample collection valve in a closedstate and a first outlet valve in a closed state; discharging thesanitizing fluid out a second outlet valve, the second outlet valvebeing located downstream of the first outlet valve; opening a samplecollection valve while the sanitizing fluid inlet valve and first outletvalve are closed; drawing a fluid sample from the enclosed containerinto a first portion of the fluid flow path and directing the fluidsample from the first portion of the fluid flow path into a variablevolume reservoir located downstream from the first portion of the fluidflow path, the variable volume reservoir comprising a reservoir inlet, areservoir outlet, and a reservoir having a volume that can changebetween a first volume and a second volume, with the second volume beingsmaller than the first; directing the fluid sample out of the variablevolume reservoir by changing the reservoir volume from the first volumeto the second volume, and into a second portion of the fluid flow path;and discharging the fluid sample out of the first outlet valve while thesanitizing fluid inlet valve and sample collection valve are closed,wherein the variable volume reservoir comprises a pump that isconfigured to draw the fluid sample into the variable volume reservoirthrough the reservoir inlet and direct the fluid sample out of thevariable volume reservoir through the reservoir outlet.
 19. The methodof claim 18, wherein after discharging the sanitizing fluid but beforedrawing the fluid sample, the method further includes: opening a gasinlet valve and directing a gas downstream through the fluid flow pathpast the sample collection valve in a closed state and through the firstoutlet valve in an open state; and discharging the gas through thesecond outlet valve to purge the sanitizing fluid from at least thefirst and second portions of the fluid flow path.
 20. The method ofclaim 18, wherein the pump comprises a diaphragm pump.
 21. The method ofclaim 20, wherein the diaphragm pump comprises a diaphragm member and ahousing with a first and second portion, the method further comprisingdrawing the fluid sample into the variable volume reservoir by movingthe diaphragm member so that a first surface of the diaphragm membercontacts the first portion of the housing.
 22. The method of claim 21,the method further comprising directing the fluid sample out of thevariable volume reservoir by moving the diaphragm member so that asecond surface of the diaphragm member contacts the second portion ofthe housing.
 23. The method of claim 21, wherein the housing comprises afirst half and a second half, the first and second halves of the housingbeing generally opposite one another.
 24. The method of claim 21,wherein the housing is a spherical housing.
 25. The method of claim 23,wherein the first surface of the diaphragm member is generally oppositethe second surface of the diaphragm member.
 26. The method of claim 25,wherein directing the fluid sample out of the variable volume reservoircauses the second surface of the diaphragm member to contact the secondportion of the housing, the diaphragm member being generally invertedwhen contacting the second portion, relative to its orientation whencontacting the first portion.
 27. The method of claim 18, wherein thesanitizing fluid comprises steam.